Microbial pathogens have evolved numerous virulence factors that are used to circumvent protective immune responses within their hosts. Many bacterial pathogens use specialized virulence protein secretion pathways to regulate innate immune responses in infected eukaryotic host cells such as macrophages. Secretion of virulence proteins from bacteria into infected macrophages often results in death of the host cell. There are several different modes by which infected macrophages can die, and the characteristics of cell death can have profound effects on the development of a protective immune response. A specialized protein export pathway known as type III secretion (T3S) allows Gram-negative bacterial pathogens to deliver effector proteins across the plasma membrane of the macrophage, with the goal of usurping or disrupting eukaryotic signaling response pathways. T3S can induce different forms of cell death in macrophages infected with Gram-negative bacteria. T3S can induce a non-inflammatory form of cell death known as apoptosis in macrophages, and this form of cell death is important for virulence of several bacterial pathogens. Alternatively, macrophages contain surveillance mechanisms that allow them to sense T3S as a danger signal, resulting in activation of a serine protease called caspase-1, and elicitation of inflammatory cytokines and protective immune responses against pathogens. Pathogenic Yersinia species can use T3S and the YopJ effector protein to inhibit the NF-:B signaling pathway and induce apoptosis in macrophages. A novel isoform of YopJ (YopJKIM) was discovered in Yersinia pestis, which can activate apoptosis and caspase-1, promoting release of cytokines from infected macrophages, a process referred to as inflammatory apoptosis. This is the first example in which a T3S effector protein is sensed as a danger signal, resulting in caspase-1 activation in macrophages. The above finding raises several key questions: How does the YopJKIM protein differ from canonical YopJ proteins that do not activate caspase-1?;What signaling pathways in the macrophage are required to activate inflammatory apoptosis?;Are immune responses regulated by caspase-1 protective or pathogenic with respect to Yersinia infections? To answer these questions the following specific aims are proposed: In Aim 1, the key features of YopJKIM that are required for caspase-1 activation in Yersinia-infected macrophages will be elucidated. In Aim 2, the importance of the NF-:B signaling pathway for caspase-1 activation in Yersinia-infected macrophages will be determined. In Aim 3, the importance of the apoptosis machinery for caspase-1 activation in Yersinia- infected macrophages will be determined. In Aim 4, the role of caspase-1 in protective immune responses to Yersinia in vivo will be investigated.